Project Summary/Abstract Cytosine DNA methylation is an epigenetic mark for gene silencing that is important in many gene regulatory systems including mammalian imprinting, X-chromosome inactivation, and the silencing of transposons and other DNA sequences containing either direct or inverted repeats. Methylation is important in cancer biology, as tumors often show both genome wide demethylation and hypermethylation of specific tumor suppressor genes. We are studying the mechanisms of DNA methylation control in the model plant Arabidopsis, which has methylation systems that have much in common with mammalian systems. Both forward and reverse genetics can be performed, and Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for detailed analysis. In Arabidopsis, the initial establishment of methylation requires the activity of DRM2, a homolog of mammalian Dnmt3, which is guided to appropriate targets, at least in part, by small interfering RNAs. Once established, DNA methylation is maintained by three different systems for methylation of cytosines in three different sequence contexts, CG, CHG and CHH (asymmetric). CG methylation is maintained by MET1, a homolog of mammalian Dnmt1. CHG methylation is maintained by the CMT3 DNA methyltransferase, which is guided to appropriate targets by the methylation of histone H3. CHH methylation is maintained by the persistent activity of DRM2, which is targeted by small interfering RNAs. In this proposal, we plan to further study these DNA methylation control mechanisms. First, we plan to study the poorly understood process by which DNA methylation is initially established, a process called de novo methylation. We have developed assays that allow us to perform both reverse and forward genetic screens to identify genes required for proper de novo DNA methylation. These mutant screens have already yielded several interesting genes, and a major part of this proposal is to perform a detailed molecular genetic characterization of these genes to understand more about the mechanism by which they contribute to de novo methylation. We also propose to continue additional genetic screens, and to characterize a set of 35 new mutants we have isolated that block de novo methylation. The second major aim of this proposal is to further study the mechanisms by which CG DNA methylation is maintained, by studying a new factor called UHRF1 in mammals and ORTH/VIM in Arabidopsis, which appears to work by binding directly to previously methylated CG sites and recruiting the CG methyltransferase to further maintain methylation. We propose to study the function of six Arabidopsis ORTH/VIM genes by using loss of function genetic alleles, understanding the specificity of chromatin interactions for each gene, and performing molecular and genetic tests of the function of the different domains of each protein. These studies should shed new light on DNA methylation systems in Arabidopsis and have implications for related systems in mammals and other eukaryotes.